Microarray Work Process.

This chapter deals with overall view at the microarray work process and tools used for each particular task within the work process. We recommend to familiarize yourself with it prior to starting microarrays operations.

Work Process Concepts.

Microarray manufacturing and processing divided into stages. Each stage consists of a number of consecutive steps that are normally done by one person and have to be completed without long interruptions between them. A stage itself is an accomplished portion of work that does not require immediate transition to the next stage in the process. Different stages can be accomplished by different specialists. Results of each process stage obtained in the form of a material entity (chemical solution, microarray slide etc.) or in the form of a document (paper document, computer file, computer data repository entry) can be saved or stored for a relatively long period of time and in some cases reused. Each consecutive stage in the microarray processing uses results of one ore more previous stages a prerequisite.

The suit of LabNEXT tools called Xpressway combines robotics, software and instructions designed in the way that supports stage-by-stage progress through the microarray work process. There is a dedicated tool or document intended to automate work in each stage or to guide through the stages where automation is not possible. All tools can operate as a complex. There are means of passing information between them in the form of computer files or unified data repository.

LabNEXT uses the data repository to store information that is used by LabNEXT proprietary software and systems only and where using of alternative programs by other vendors is not expected. Computer files as a way of data communications used where is an option of using tools other than from LabNEXT exists.

The following table provides complete catalogue of stages and steps of the Microarray production and processing work flow and specifies the tool or instruction provided by LabNEXT to be used in a particular stage.

Stage	Steps	Prerequisites	Tools or Instructions Provided by LabNEXT	Results	Comments	Approx. Duration.
1. Prepare DNA clone library.	Amplify DNA clones Perform DNA purification. Control amount of DNA amplified. Record annotation of DNA clones in the storage containers (tubes or microplates)	No.	Documents: LabNEXT Glass Microarray Protocols. Use to obtain protocol of amplification.	Tubes or microplates with DNA clones in the amount sufficient for at least one print. Document with annotation of clones in microplates or tubes.	If this stage has done by an external facility it is essential to OBTAIN THE ANNOTATION DOCUMENT of the clones provided for printing.	1 day per 300 clones.
2. Select type of microplates for printing.	Register geometrical parameters of microplates that will be used in the microarrayer.	No.	Program: JobWizard (Step 2). Use to register geometrical parameters of microplates.	Data repository record with plates specifications.	The result of this activity can be reused. Once registered and saved in the data repository a particular type of microplate can be used in future with no need to re-enter the data. Each new type of microplates requires separate registration.	1 hour
3. Transfer DNA clones in the source microplates.	Transfer DNA clones form the library containers to the source microplates. Dry DNA clones. Record annotation of the source plates. Specify the source plates that will be used for a particular Microarray.	Annotation document of the DNA library. Source microplates specification.	Program: JobWizard (Steps 3 and 4) Use to record positions of clones in the source microplates.	Data repository records with source plates annotations. Source plates with dry DNA clones.	The results of this activity can be reused. If there is a possibility to print more than one time from a microplate annotation of this microplate can be retrieved from the repository. Accurate annotation of source plates is an essential factor in obtaining credible experimental data.	1 hour per 384 well microplate.
4. Define the type and the pattern of printing needles.	Specify type of printing needles. Specify position of the needles in the printing head. Record head configuration.	Repository records with geometrical dimentions and physical parameters of printing needles. (Provided by LabNEXT)	Program: JobWizard (Step 4)	Repository record deals with printing head configuration.	The results of this stage are reusable. Once recorded to the repository a particular head configuration can be used for printing different microarrays.	1 hour
5. Design physical layout of microarray and pre-spotting slides.	Specify geometrical parameters of substrates. Specify number of repetitions of printed spots. Specify position of the clones on the substrate surface. Specify	Annotated source plates records. Printing head configuration record.	Program: JobWizard (Step 5-6) Document:: Microarray Geometry	Microarray layout record. Pre-spotting slide layout record.	The results of this activity can be reused.	2 hours
6. Prepare system for printing.	Specify number of microarrays to be printed. Specify location of microarray substrates and pre-spotting substrates on the work board of the robot. Save parameters of the Print Job in the data repository. Turn on washer and dryer. Place substrates on the work board. Insert printing needles in the printing head Dissolve dried DNA in the source microplates. Place source microplate on the work board.	Repository records of all previous stages. Printing buffer. Source plates. Microarrays substrates. Pre-spotting substrates.	Program: JobWizard (Step 7-8) Documents: LabNEXT Glass Microarray Protocols. Documents: LabNEXT Glass Microarray Protocols. Use to obtain information deals with preparation of the printing buffer.	Repository record with Print Job parameters.	The Print Job can be launched multiple times if more than 13 microarrays required. In this case it is necessary to reload the microarrayer with new slides and launch the print job again. If more then one microarrayer work with one to the repository all of them can perform a particular print job simultaneously.	3 hours
7. Print microarrays.	Launch microarrayer.	Repository record with Print Job parameters.	Program: JobControl	Microarrays. Microarray Annotation file. (A computer file in TXT format that contains annotation of the spots on the microarray surface)	The annotation file will be used for microarray image processing.	1 hour per 384 well plate with 4 needles
8. Perform post printing chemical treatment of microarrays.	Block active surface Wash microarrays.	Printed microarrays.	Documents: LabNEXT Glass Microarray Protocols.	Microarrays ready for hybridization.	The processed microarrays can be stored for a long time.	4 hours
9. Hybridize microarrays.	Prepare hybridization probes Perform pre-hybridization. Perform hybridization. Wash microarrays.	Microarrays ready for hybridization.	Documents: LabNEXT Glass Microarray Protocols.	Microarrays ready for scanning.		1 day
10. Scan microarrays.	Adjust scanning parameters to equalize signals in scanners’ channels. Scan microarrays. Name and save image files.		Program: Xplore Use for measurement s of integral intensity of microarrays surface	Microarray image files. (16-bit TIFF computer files.) One per each channel.	It may be necessary to perform multiple scans in order to analyze surface intensity and readjust scanner sensitivity. Typically it takes 3-5 iterations to obtain well balanced images in both channels. LabNEXT does not provide scanning equipment.	1 hour
11. Extract image data.	Locate DNA spots on the microarray surface. Extract data and save intensity data file.	Microararys image files. (one per each channel) Microarray annotation file.	Program: Xplore Use for location of the spots and extraction of intensity data.	Integral intensity file. (A computer file in TXT format that contains data of intensity of all spots )	The integral intensity file will be used by the Xplain image analysis program.	1 hour per microarray
12. Analyze Microarray Data	Preview differential expressions data and quality control statistics. Define critical signal to noise ratio and select clones that produces credible results. Save report file.	Integral intensity file.	Program: Xplain Document:: Xplain Use to obtain instructions regarding interpretation of the experimental data.	Report File (A computer file in TXT format that contains differential expression data for each selected clone form the Microarray.)	The Report file can be used for publications or processing using external software.	10 min per microarray.